A major recent advancement for the field of pain research is the recognition of immune system dysregulation as a contributor to the most serious adverse consequences of injury. Accumulating data from clinical and laboratory studies place the activation of B lymphocytes at the center of much of this work, particularly with respect to chronic pain and disability-related outcomes. Validation of this B cell hypothesis could lead directly to trials testing the efficacy of novel or existing immunomodulating agents on posttraumatic pain. Key goals and outcomes of peritraumatic B cell modulation reflected in our experimental design include enhancing the rate and improving the quality of recovery while reducing chronic pain, functional loss and neuropsychiatric consequences such as anxiety and cognitive decline. To achieve our goals a well-validated core mouse model of limb fracture will be employed with additional studies to be conducted in incisional and nerve injury models to broaden our assessment of B cell mediated effects. Age and sex will be included as variables to enhance rigor. Often discussed but seldom achieved translation of the studies to a larger species (dogs) and finally to humans will be achieved in this innovative project. In the project's first aim we rigorously address the role of B cell activation and autoantibody formation in pain, functional and neuropsychiatric outcomes after fracture injury in mice. The tools used directly (anti- CD20) and indirectly (sympatholysis, anti-IL-6) modulate B cell function to mimic potential therapeutic approaches. The outcomes selected are highly robust and quantify behavioral, cellular and biochemical features of recovery from trauma. In the second aim we assess the contributions of B cell activation and autoantibody formation on pain, function and neuropsychiatric outcomes in models of soft tissue injury (incision) and nerve damage (selective ligation). These experiments will help us define the breadth of B cell effects by examining specific contributors to trauma-related pain. In the project's final aim, we make the critical transition to examination of autoantibody formation in dogs undergoing tibial surgery and in humans undergoing knee replacement involving two unique collaborations. Both are common surgeries with substantial rates of persistent pain and disability. Preliminary evidence suggests that pain-promoting and function-limiting antibodies are formed in 40-50 percent of these cases. Chronic pain syndromes after injuries and surgery have substantial functional, neuropsychiatric and financial consequences. We do not have effective non-addictive therapies to prevent or treat posttraumatic pain in disease-modifying fashion. If this B lymphocyte target validation effort is successful, the door will be open to the design of definitive clinical trials using novel or existing immunomodulating tools.